The Notch signal transduction pathway controls cell fate decisions important for neurogenesis and hematopoesis, and is misregulated in human cancers. Signaling results in the proteolytic release of the intracellular domain (ICD) of the Notch receptor, a dedicated transcriptional co-activator of CSL enhancer-binding proteins. Our lab recently developed a chromatin-based system that recapitulates Notch transcription in vitro. We show that the ICD lacks an intrinsic activation domain, but rather recruits Mastermind (MAM), which provides two distinct regulatory domains. A MAM chromatin-specific activation domain recruits the CBP/p300 histone acetyltransferase. The second activation domain, which is essential for Notch transcription in vivo, also serves to couple Notch transcription to modification and proteolytic turnover of the ICD within the Notch enhancer. This region of MAM also mobilizes CBP/p300 to nuclear inclusion bodies similar to those formed by mutant polyglutamine-repeat proteins in neurodegenerative diseases. These findings indicate that MAM is a novel transcriptional regulator which functions to couple activation to turnover, ensuring that the Notch enhancer complex is short-lived. We plan to further define the Notch regulatory mechanism through the following aims: 1) Analyze the mechanisms that regulate initiation and elongation of Notch transcription on chromatin templates and identify the protein(s) that interact with the MAM activation domains; 2) Assess the process by which MAM induces the formation of nuclear inclusion bodies, determine the composition of these structures and assess connections to the ubiquitination-proteolysis machinery; 3) Analyze the role of MAM in the proteolytic turnover of the Notch ICD. We will identify the nuclear kinase that phosphorylates the ICD and determine if the Sel-10 ubiquitin ligase is recruited to the Notch enhancer; and 4) Characterize the structure and mode of regulation of a natural (periodic) Notch enhancer in the cell-free in vitro transcription system. These studies will help elucidate the Notch transactivation mechanism and its role in the modification and destruction of the ICD, and may suggest approaches for blocking Notch signaling in human cancers.